UDP-glucose, cereblon-dependent proinsulin degrader

Insulin secretion is regulated in multiple steps, and one of the main steps is in the endoplasmic reticulum (ER). Here, we show that UDP-glucose induces proinsulin ubiquitination by cereblon, and uridine binds and competes for proinsulin degradation and behaves as sustainable insulin secretagogue. Using insulin mutagenesis of neonatal diabetes variant-C43G and maturity-onset diabetes of the young 10 (MODY10) variant-R46Q, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) protects cereblon-dependent proinsulin ubiquitination in the ER. Cereblon is a ligand-inducible E3 ubiquitin ligase, and we found that UDP-glucose is the first identified endogenous proinsulin protein degrader. Uridine-containing compounds, such as uridine, UMP, UTP, and UDP-galactose, inhibit cereblon-dependent proinsulin degradation and stimulate insulin secretion from 3 to 24 h after administration in β-cell lines as well as mice. This late and long-term insulin secretion stimulation is designated a day sustainable insulin secretion stimulation. Uridine-containing compounds are designated as proinsulin degradation regulators.

(S1b and c) Intracellular proinsulin and UGGT1 were analyzed by WB (c and Fig. S1b).
WT-insulin binds to UGGT1, and is retained in the ER. Arginine administration releases proinsulin from UGGT1 in a competitive manner.
On the other hands, R46Q proinsulin binds to UGGT1 like WT-proinsulin in the absence of 4 arginine. Arginine administration has no effect on the R46Q and UGGT1 interaction, leading to less induced R46Q secretion by arginine.  (S3a) Higher amount of secreted insulin K88R with/without arginine.
(Healthy) WT-proinsulin; in the absence or low concentration of arginine, WT-proinsulin binds to UGGT1 mainly. Arginine administration releases proinsulin from UGGT1. Free proinsulin partially binds to cereblon and DDB1 for ubiquitination, and the rest free proinsulin will be secreted.
(Neonatal diabetes) C43G-proinsulin; in the absence and presence of arginine C43G binds mainly to CRBN and DDB1 and is ubiquitinated. (d2) Arginine administration triggers proinsulin releasing from UGGT1 and HRD1 complex.
(d5) A half of free proinsulin binds to CRBN and DDB1 with unknown endogenous ligand X, and is ubiquitinated and degraded. (f1) In the absence or low concentration of arginine, proinsulin binds to UGGT1 and HRD1.

Supplementary
(S5a) Cereblon bound to UGGT1 directly in vitro, and UDP-glucose induced the interaction between cereblon and UGGT1.
(S5d) UGGT1-WT and GTF mutant bound to UTP and glucose, although UGGT1-3W>3A mutant protein did not bind to neither UTP nor glucose.